Platelets are the smallest blood cells with primary function of coagulation and hemostasis. When a body gets injured to bleed, platelets will gather at the site of injury in droves within several seconds. Firstly the platelets release vasoconstrictive substance for constricting the damaged blood vessel in varying degrees, then the platelets and other blood-clotting substance in the blood adhere to the damaged vessel wall and clump together to form a clot and accordingly block the damaged wounds and blood vessels. The platelet count of a healthy person is 100×109/L˜300×109/L and the platelet's average life is 8˜12 days. Thrombopenias means that the platelet count results are below a lower limit of reference value due to various reasons.
Thrombopenias may cause the following harmfulness: 1. causing mucosal bleeding (such as nasal mucosa bleeding, oral mucosal bleeding, gastrointestinal mucosal bleeding, genitourinary tract bleeding, vaginal bleeding, etc.); 2. postoperative massive hemorrhage; 3. multiple petechiae and purpura occurred most frequently in the legs; 4. causing gastrointestinal massive hemorrhage and central nervous system internal hemorrhage that may threaten life.
Thrombopenia is caused by a great many reasons, such as decrease in platelets generation, excess destruction of platelets, excess retention of platelets within the spleen, etc. (1) Decrease of platelets generation is caused by destruction of hematopoietic stem cells or affection of their proliferation in the bone marrow cells due to some factors such as drugs, cancer, infection, ionizing radiation, etc. (2) Excess destruction of platelets may commonly be found in idiopathic thrombocytopenic purpura and consumptive thrombocytopenia, such as disseminated intravascular coagulation, thrombotic thrombocytopenic purpura. (3) Excess retention of platelets within the spleen may commonly be found in hypersplenism. The above factors often coexist on one body. In clinical, except that pseudo-thrombocytopenia doesn't need treating, both drug-induced thrombocytopenia and pathological thrombocytopenia need treating by treatment means.
Drug-induced thrombocytopenia (DITP) is a hemorrhagic disease resulting from the decrease of platelet count in the blood due to some drugs, and manifests that the drug-induced platelet count is lower than 100×109/L and the platelet count of severe thrombocytopenia is lower than 5×109/L. DITP is often caused by the following drugs: anti-clotting drugs such as heparin, antineoplastic drugs and immunosuppressant, antibacterial drugs such as chloramphenicol and sulfonamides, antipyretic analgesics such as aspirin and acetaminophen, diuretics such as chlorothiazide, antiepileptic drugs such as phenytoin and carbamazepine, hypoglycemic drugs such as chlorpropamide and tolbutamide, oestrogenic substance such as diethylstilbestrol, some vaccines, some Chinese medicine preparations, some lipid-lowering drugs, cimetidine, bismuth, digitoxin, organic arsenics, and other drugs.
The pathological thrombocytopenia mainly includes primary and secondary thrombocytopenic purpura, aplastic anemia, acute leukemia, megaloblastic anemia. DIC, hypersplenism, radiation syndrome, kala azar, typhoid fever, tuberculosis, bone metastases, and progressive extracorporeal circulation and the like. The most common in clinical is thrombocytopenic purpura.
For many years, many scholars at home and abroad conduct in-depth research on the treatment of Thrombocytopenia, and have achieved some achievements. There are a plurality of methods for treating Thrombocytopenia, comprising first-line treatment: administration of glucocorticosteroid, intravenous immunoglobulin and splenectomy; second-line treatment: intravenous anti-Rh(D) immunoglobulin and immunosuppressant. The treatment methods, such as platelet transfusion, plasma exchange, protein immunoadsorption, can be used if the above first and second line treatments have failed. Wherein, the platelet transfusion is an effective method to treat Thrombocytopenia, but its clinical application is limited because of short preservation time, lack of blood supply, high cost, possible blood-borne infections, transfusion reaction and producing platelet antibodies. In addition, repetition of platelet transfusions may, cause platelet transfusion refractoriness, therefore threatening the patient's life all the time. Although interleukin-11 (IL-11) and thrombopoietin (TPO) have preferable curative effect for treating Thrombocytopenia, they are unable to be widely applied in clinic due to great adverse reactions and expensive price. Therefore, it is a goal to research and develop a safe and effective therapeutic drug which has significant social benefits and broad market prospects.
With respect to Thrombopenia, there are also treatment theories in Traditional Chinese medicine (TCM) theory. For thrombocytopenic purpura (1TP), commonly used therapeutic drug are platelet increasing capsules, Weixuening particles, etc. For thrombocytopenia caused by chemotherapy, TCM considers chemotherapeutic drugs is a drastic drug with pyretic toxicity, and they can consume qi and impair yin, and damage zang-fu viscera such as spleen, kidney and liver, thereby exhausting congenital and postnatal source and resulting in injury of qi and blood, deficiency of liver and kidney, hypofunctioning of spleen and stomach, etc. The most commonly used Traditional Chinese Medicine are Radix et Rhizoma Ginseng, Astragali Radix, Rhizoma Atractylodis Macrocephalae, Angelicae Sinensis Radix, Rehmanniae Radix, Asini Corii Colla, Spatholobi Caulis, Psoraleae Fructus, Lycii Fructus, Ligustri Lucidi Fructus, Agrimoniae Herba, Arachis hypogaea Linn, etc., as well as some compound preparations such as Compound Zaofan pills, and Sheng Ban Recipe.
Radix et Rhizoma Notoginseng, aslo known as Panax notoginseng or Stephania sinica Diels, is dried roots of panax notoginseng (Burk.) F. H. Chen of Panax of araliaceae. As written in The Compendium of Materia Medica, “Panax notoginseng has stypticity and analgesia effects, and can stop uncontrollable bleeding due to sword cuts, arrow wounds, falling injury, flogging injury and sore by applying the chewed or powdered Panax notoginseng onto the wounds. Panax notoginseng can also treat diseases such as hematemesis, dysentery characterized by blood in the stool, endometrorrhagia, persistent menstruation, postpartum retention of lochia, dysmenorrheal due to poor blood circulation, swelling and pain of eyes, and tigerbite and snakebite injury”. Referring to The Records of Combination between Traditional Chinese Medicine and Western Medicine. “Panax notoginseng can dissipate extravasated blood without hurting new blood, which is a marvelous drug for regulating blood.” As referring to New Compilation of Materia Medic), “Roots of panax notoginseng are miracle cures for stopping bleeding”. Chinese Pharmacopoeia defines the major function of Radix et Rhizoma Notoginseng as “dissipating stasis and stopping bleeding, diminishing swelling and relieving pain”. Radix et Rhizoma Notoginseng is widely applied to treat internal and external hemorrhage syndrome caused by traumatic injury and internal injury for thousands of years.
Dencichine (also called Neurotokin, chemical name: β-N-oxalyl-L-α, β-diaminopropionic acid, referred to as ODAP), is a non-protein amino acid, and is hemostatic active ingredient of Radix et Rhizoma Notoginseng. Rao et al isolated dencichine from a seed of Lathyrus sativus and identified its chemical constitution in 1964, and successfully synthesized the dencichine in 1971, and synchronously researched the relation between the optical activity and central toxicity of dencichine. Hereafter, various synthetic methods were successively reported. At present, the research on central neurotoxicity of Dencichine has gone deep into cellular and molecular level, and it is considered that the central neurotoxicity of dencichine is because that it is an analogue of L-Glutamate which polarizes central nervous system cell membrane and affects activity of ions including Na+, K+, Ca2+, etc.
In the middle of 1980s, Chinese medicine workers started to research synthesis, pharmacology and toxicology of dencichine. In 1984, Zhao Guoqiang et al., worked in Tianjin Institute of Traditional Chinese Medicine, synthesized dencichine which is the active hemostatic ingredients of Notoginseng Radix et Rhizoma, and researched the effect of dencichine and its enantiomorph of β-N-oxalyl-D-α, β-diaminopropionic acid on hemostasis, increase of platelet count and neurotoxicity, etc., thus finding out both dencichine and the enantiomorph thereof have a significant effect on the hemostasis and increase of platelet count, etc. Zhao Guoqiang dissolved 1 mg dencichine into 0.5 ml Ringer-Locke solution to prepare a mixed solution, and injected the mixed solution into abdominal cavity of a female mouse, and it was found that dencichine is able to markedly increase the platelet count by 30%. In 1988, Lu Qi et al of Jilin Agricultural University respectively separated dencichine used as hemostatic ingredient from Traditional Chinese Medicine such as Ginseng Radix et Rhizoma, whereby scientifically verifying that Traditional Chinese Medicine such as Ginseng Radix et Rhizoma etc. have hemostasis effect, which was recorded in ancient prescription. Liu Hezhi et al have researched hemostasis mechanism of dencichine, and considered that injection of Notoginseng Radix et Rhizoma could make thrombocyte generate viscous deformation movement such as stretch, pseudopodia, aggregation, deformation, as a result some cells were broken down or dissolved, and degranulation and secretory response was caused, thereby inducing thrombocyte to release hemostatic active substance such as ADP, platelet factor and calcium ion, so as to achieve the hemostasis purpose.
With respect to the report that dencichine can increase the platelet count, we carried out experimental research on dencichine for use in treatment of Thrombocytopenia. The results show that it has no obvious change on the platelet count of rabbits by using single-dose injection of dencichine and various doses of drug respectively (see Table 1).
TABLE 1Influence of Dencichine for injection on the plateletcount of rabbits(n = 6, mean ± SD)DosePLT(×109/L)Group(mg/kg)0 min30 min60 minSodium—529.0 ± 54.2512.8 ± 54.7 524.8 ± 49.9chlorideinjectionReptilase ®0.3 613.0 ± 118.8488.5 ± 141.9519.2 ± 90.5KU/kgDencichine1.00 491.2 ± 163.1526.5 ± 143.8 541.0 ± 121.6forinjection0.20574.0 ± 64.3502.3 ± 60.8  518.2 ± 128.50.04523.3 ± 78.5451.0 ± 144.5504.0 ± 87.8
In the repeated prophylactic administration process, prophylactic administration for 3 days, 5 days and 7 days respectively, the results show that prophylactic administration of interleukin 11 or dencichine for 5 days and 7 days respectively increase the platelet count (compared with that of model group). The platelet count had no significant difference compared with that of model group by prophylactic administration for 3 days (Table 2).
TABLE 2Influence of dencichine with different administrationtime on platelet count of rats with thrombocytopeniacaused by carboplatin (x ± sD, ×109/L)Injection ofProphylacticCarboplatinInitial valueadministrationfor 10 daysBlank group747.0 ± 106.88635.7 ± 118.11 788.0 ± 147.35Model group716.6 ± 62.57 651.3 ± 97.93 115.9 ± 12.71Prophylactic732.3 ± 197.06860.5 ± 113.92179.5 ± 26.76administration ofinterleukin for7 daysProphylactic743.7 ± 226.92772.8 ± 70.63#166.2 ± 18.20administration ofdencichine for7 daysProphylactic724.0 ± 178.87759.5 ± 145.34178.3 ± 57.08administration ofinterleukin for5 daysProphylactic736.2 ± 182.37773.2 ± 142.02176.2 ± 28.34administration ofdencichine for5 daysProphylactic736.3 ± 175.56820.7 ± 155.13 88.5 ± 51.49administration ofinterleukin for3 daysProphylactic717.5 ± 276.94656.3 ± 67.35 110.2 ± 40.43administration ofdencichine for3 days
Pharmacodynamic study further finds that dencichine has a significant effect against decrease of a rat's platelet count caused by carboplatin, which shows the effect for increasing thrombocyte, and simultaneously improving the function of thrombocyte to fight against decrease of thrombocyte aggregation rate of the rat caused by injecting carboplatin, thus increasing thrombocyte aggregation rate. So far, it has been reported that a little dencichine can be used as hemostatic drug in prior art, as disclosed in the patent application with publication No. CN1292376A that administrating a little dencichine can achieve hemostasis. However, it has never been reported that dencichine is able to treat Thrombocytopenia.